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. 2018 Aug:521:99-107.
doi: 10.1016/j.virol.2018.05.028. Epub 2018 Jun 11.

Live-attenuated bivalent measles virus-derived vaccines targeting Middle East respiratory syndrome coronavirus induce robust and multifunctional T cell responses against both viruses in an appropriate mouse model

Bianca S Bodmer  1 , Anna H Fiedler  1 , Jan R H Hanauer  2 , Steffen Prüfer  2 , Michael D Mühlebach  3
Affiliations

Live-attenuated bivalent measles virus-derived vaccines targeting Middle East respiratory syndrome coronavirus induce robust and multifunctional T cell responses against both viruses in an appropriate mouse model

Bianca S Bodmer et al. Virology. 2018 Aug.

Abstract

Cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to occur, making it one of the WHO´s targets for accelerated vaccine development. One vaccine candidate is based on live-attenuated measles virus (MV) vaccine encoding the MERS-CoV spike glycoprotein (MERS-S). MVvac2-MERS-S(H) induces robust humoral and cellular immunity against MERS-S mediating protection. Here, the induction and nature of immunity after vaccination with MVvac2-MERS-S(H) or novel MVvac2-MERS-N were further characterized. We focused on the necessity for vector replication and the nature of induced T cells, since functional CD8+ T cells contribute importantly to clearance of MERS-CoV. While no immunity against MERS-CoV or MV was detected in MV-susceptible mice after immunization with UV-inactivated virus, replication-competent MVvac2-MERS-S(H) triggered robust neutralizing antibody titers also in adult mice. Furthermore, a significant fraction of MERS CoV-specific CD8+ T cells and MV-specific CD4+ T cells simultaneously expressing IFN-γ and TNF-α were induced, revealing that MVvac2-MERS-S(H) induces multifunctional cellular immunity.

Keywords: Antibody responses; MERS Coronavirus; Measles Virus; Multifunctional T cells; Vaccine platform.

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Figures

Fig. 1
Fig. 1
Generation, characterization, and immunogenicity of MVvac2-MERS-N (A) Immunoblot analysis of Vero cells infected at an MOI of 0.03 with MVvac2-MERS-N(P), MVvac2-MERS-N(H), or MVvac2-GFP(N) as depicted above the lanes. Uninfected cells served as mock control. Blots were probed with rabbit serum reactive against MERS-CoV (upper blot) or mAb directed against MV-N (lower blot). (B, C) Growth kinetics of recombinant MV on Vero cells infected at an MOI of 0.02 with MVvac2-MERS-N(P), MVvac2-MERS-N(H), or MVvac2-GFP(P). Virus from (B) supernatant or (C) cell-associated virus was harvested at indicated days post infection (dpi) and titrated on Vero cells. Means and standard deviation from three independent experiments. (D) Vaccination scheme for IFNAR-/--CD46Ge mice immunized with respective recombinant MV. (E, F) Analysis of virus neutralizing titers (VNT) in vaccinated animals on day 49 post prime immunization for complete neutralization of (E) 200 TCID50 of MERS-CoV or (F) 50 PFU of MV. Medium inoculated mice served as mock control. VNTs were calculated as the highest dilution abolishing infectivity. Dots represent single animals (n = 6); horizontal lines represent mean per group. The y-axis starts at the detection limit; all mice at the detection limit had no detectable VNT. (G) Secretion of IFN-γ after antigen-specific re-stimulation of splenocytes harvested 32 days post prime immunization and after co-culture with JAWSII (left) or DC2.4 (middle) dendritic cells transgenic for MERS-N (black) or untransduced controls (NC, white). (Right) To analyze cellular responses directed against MV, splenocytes were stimulated with 10 μg/mL MV bulk antigen (MV bulk) or left unstimulated (sham). The reactivity of splenocytes was confirmed by ConA treatment (10 μg/mL). TCID50, tissue culture infectious dose 50; One-way-ANOVA with Tukey multiple comparison. *: p < 0,05; **: p < 0,01; ***: p < 0001; ****: p < 0,0001.
Fig. 2
Fig. 2
Humoral and cellular immune responses induced by live or UV-inactivated MV-MERS-S(H). (A) Schematic depiction of vaccination scheme for IFNAR-/--CD46Ge mice vaccinated with MVvac2-MERS-S(H), UV-inactivated MVvac2-MERS-S(H), or MVvac2-ATU(P) as vector control. (B-G) Virus neutralizing titers (VNT) in vaccinated mice. Titers of (B, D, F) MV or (C, E, G) MERS-CoV neutralizing antibodies in sera of (B, C) naїve mice, or in sera of mice after (D, E) prime- or (F, G) boost-immunization. One-way ANOVA with Tukey multiple comparison. * : p < 0,05; * *: p < 0,01; ***: p < 0001; ****: p < 0,0001. (H) Secretion of IFN-γ after antigen-specific re-stimulation of splenocytes. IFN-γ ELISpot analysis using splenocytes of mice vaccinated on days 0 and 28 with indicated vaccines isolated 21 days after boost immunization and after incubation with indicated stimuli (MERS-S peptide S1165, MV bulk antigen (MV bulk), immunodominant ovalbumin-derived SIINFEKL-peptide (SIN) as a peptide negative control) or untreated (mock). The reactivity of splenocytes was confirmed by Concanavalin A (ConA) treatment (10 μg/mL). The number of cells per 1 × 106 splenocytes represent the amount of cells expressing IFN-γ upon re-stimulation. Dots represent individual animals, horizontal bars mean. One-way ANOVA with Tukey multiple comparison. ****: p < 0,0001.
Fig. 3
Fig. 3
Characterization of T-cell responses against MERS-CoV in older mice. (A) Schematic depiction of vaccination scheme for at least 7 months old IFNAR-/--CD46Ge mice vaccinated with MVvac2-MERS-S(H) with splenocytes harvest 21 days after boost. (B) These splenocytes were re-stimulated with MERS-S peptide S1165, MV bulk Ag (MV bulk), ovalbumin-derived SIINFEKL-peptide (SIN) as peptide negative control, medium (mock), or Concanavalin A (ConA) as a general T-cell activator positive control. Dots represent individual animals, horizontal bars mean. One-way-Anova with Tukey multiple comparison. *: p < 0,05; ****: p < 0,0001.
Fig. 4
Fig. 4
Detection of multi-functional T-cell responses induced by vaccination with MVvac2-MERS-S(H). Harvested splenocytes of MVvac2-MERS-S(H) vaccinated mice (same as depicted in Fig. 3) were re-stimulated and subjected to intracellular staining (ICS) for IFN-γ and TNF-α and stained for extracellular T-cell markers CD4 and CD8 for flow cytometry analysis. (A - C) Gating strategy for analysis of CD8+ or CD4+ T-cells expressing IFN-γ or TNF-α within splenocytes stimulated with (B) S1165 peptide or (C) MV-bulk Ag. Duplicates (not shown) and dead cells (A) were excluded from analysis. (B, C) CD8+ and CD4+ cells were separately subjected to analysis for IFN-γ- (left panels), TNF-α- (middle panels) or double-positive cells (right panels). Quantification of flow cytometry data of (D) CD8+- and (E) CD4+-positive cells after incubation with indicated stimuli (MERS S-specific peptide S1165, MV bulk Ag (MV bulk), immunodominant Ovalbumin-derived SIINFEKL-peptide (SIN) as a peptide negative control, or untreated cells (mock); reactivity of splenocytes was confirmed by Tetradecanoylphorbol-acetate and Ionomycin (TPA/Iono) treatment (10 μg/mL). Dots represent individual animals, horizontal bars mean. Repeated-measures one-way ANOVA with Tukey multiple comparison. *: p < 0,05.
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